Persoonia 38, 2017: 38–57 www.ingentaconnect.com/content/nhn/pimj RESEARCH ARTICLE http://dx.doi.org/10.3767/003158517X693174 Taxonomy of Tricholoma in northern Europe based on ITS sequence data and morphological characters J. Heilmann-Clausen1, M. Christensen2, T.G. Frøslev 3, R. Kjøller 4 Key words Abstract Based on molecular and morphological data we investigated the taxonomy and phylogeny of the ecto- mycorrhizal genus Tricholoma in northern Europe. Our phylogenetic tree confirmed the presence of at least 72 Agarics well circumscribed species within the region. Of these, three species, viz. T. boreosulphurescens, T. bryogenum biogeography and T. ilkkae are described as new to science, based on morphological, distributional, ecological and molecular cryptic species data. Several other terminal branches represent putative cryptic taxa nested within classical species or species ectomycorrhizal fungi groups. Molecular type studies and/or designation of sequenced neotypes are needed in these groups, before host selection the taxonomy can be settled. In general our phylogenetic analysis supported previous suprageneric classification morphological traits systems, but with some substantial changes. Most notably, T. virgatum and allies were found to belong to sect. phylogeny Tricholoma rather than sect. Atrosquamosa, while T. focale was found to be clearly nested in sect. Genuina rather Tricholomataceae than in sect. Caligata. In total, ten sections are accepted, with five species remaining unassigned. The combination of morphological and molecular data showed pileus colour, pileipellis structure, presence of clamp connections and spore size to be rather conservative characters within accepted sections, while the presence of a distinct ring, and especially host selection were highly variable within these. Article info Received: 23 January 2015; Accepted: 12 May 2016; Published: 26 August 2016. INTRODUCTION indicated to be probable in Albomagister and Pseudotricho­ loma. Only the latter genus is known to occur in Europe, were The genus Tricholoma is a classic genus of agarics already pro- Pseudotricholoma metapodium is widespread. posed as a section by Fries (1821) and subsequently erected Tricholoma has a worldwide distribution (Tedersoo et al. 2010), as a genus by Staude (1857). but seems to be most prominent in temperate and subtropical Over the years more than 850 species epithets have been pu- zones in both the southern and northern hemisphere. All known blished or combined in the genus. Many of these have since species are known or supposed to be ectomycorrhizal (Ryberg been transferred to other genera, including Lepista, Leucopaxil­ & Matheny 2011), mainly with trees in the Pinaceae, Betulaceae lus, Lyophyllum and Melanoleuca based on various deviations, and Fagaceae, but the genus also contains species that are mainly in microscopic characters. Molecular studies have sup- associated with Eucalyptus, Dryas and Helianthemum (Bougher ported the segregation of most of these more modern genera, 1996, Christensen & Heilmann-Clausen 2013). Some species and Tricholoma in its narrow circumscription (e.g. Noordeloos form dual ectomycorrhizal and monotropoid associations linking & Christensen 1999) is supported as a monophyletic genus of trees and monotropoid plants (Leake et al. 2004). The centre ectomycorrhizal fungi within the Tricholomataceae (e.g. Mon- of species richness appears to be in North America. According calvo et al. 2002). According to Ryberg & Matheny (2012), the to Bessette et al. (2013), more than 100 species are reported genus seems to have segregated from its ancestral clade some from this continent while 63 to 88 species are listed from Eu- 60–90 million years ago in the late Cretaceous, possibly with rope (Riva 1988, Bon 1991, Kirby 2012). Several species are Pinaceae as mycorrhizal partners. A recent study (Sánchez- described or reported from Japan, New Zealand and Australia García et al. 2014) surveyed in depth the Tricholomataceae (e.g. Hongo 1988, Bougher 1996, Orlovich & Cairney 2004), but based on several molecular markers and concluded that only the overview of the species diversity in these regions is frag- the genera Albomagister, Corneriella, Dennisiomyces, Leuco­ mentary due to the lack of modern comprehensive treatments. paxillus, Pseudotricholoma, Porpoloma s.str. and Tricholoma Tricholoma species show limited microscopic variation, and are belong to the family, while other previously contained genera characterized by hyaline, subglobose to oblong spores, simple should be assigned to families, including the Lyophyllaceae pileipellis structures and lack of well-differentiated sterile ele- and a poorly resolved residual Tricholomatoid clade. Of the ments, including cystidia. Hence, species identification and genera included in the Tricholomataceae, also Porpoloma partly also the infrageneric classification has mainly been s.str. is proven ectomycorrhizal, while a biotrophic lifestyle is based on macromorphology. Singer (1986) divided the genus in four subgenera, mainly based on pileipellis structure and 1 University of Copenhagen, Natural History Museum of Denmark, Center for Macroecology, Evolution and Climate, Universitetsparken 15, DK-2100 the presence or absence of clamp connections. The four sub- Copenhagen, Denmark; genera were further divided into nine sections, of which three corresponding author e-mail: [email protected]. (Leucorigida, Iorigida and Adusta) do not belong to the genus 2 Hvidtjørnevej 1, DK-4180 Sorø, Denmark. in the current circumscription. In their treatment of the genus, 3 University of Copenhagen, Natural History Museum of Denmark, Center for Geogenetics, Øster Voldgade 5-7, 1350 Copenhagen, Denmark. Noordeloos & Christensen (1999) accepted the four subgenera 4 University of Copenhagen, Institute of Biology, Section of Terrestrial Ecol- suggested by Singer (1986), but with a more narrow definition ogy, Universitetsparken 15, DK-2100 Copenhagen, Denmark. of sections, especially in subg. Tricholoma, in which seven sec- © 2016-2017 Naturalis Biodiversity Center & Centraalbureau voor Schimmelcultures You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. J. Heilmann-Clausen et al.: Tricholoma in northern Europe 39 tions were accepted. Slightly deviating classification systems connections were explored in more detail, but many other have been proposed by other authors, including Bon (1984a, characters were described for accepted species in Christensen 1991). For a more throughout evaluation see Riva (1988) and & Heilmann-Clausen (2013). Christensen & Heilmann-Clausen (2013). Despite their attractive fruit bodies, and a long mycological tradi- Molecular & phylogenetic methods tion, the overall taxonomy in Tricholoma is still poorly resolved in DNA was extracted from dried specimens by the CTAB-chloro- Europe. Molecular data have been used to study the taxonomy form method described by Gardes & Bruns (1993). Usually, and phylogeny of some species groups (e.g. Comandini et al. one lamella was taken with a flamed pair of forceps from the 2004, Jargeat et al. 2010, Ota et al. 2012, Moukha et al. 2013), specimens. The internal transcribed spacer (ITS) region was typically resulting in the identification of cryptic diversity within amplified with the primer combination ITS1-F and ITS4 (White previously accepted species. Simultaneously, several species et al. 1990, Gardes & Bruns 1993). The PCR products were se- have been proposed in recent years without a published test quenced by Europhins Genomics (previously MWG-Biotech) or of taxonomic placement based on molecular markers (e.g. Macrogen (www.macrogen.com). Forward and reverse strands Kalamees 2001, Musemeci & Contu 2008, Ferrarese & Zaf- were sequenced using ITS1F or ITS5 (White et al. 1990) and falon 2010, Ludwig 2012), adding to taxonomic confusion in ITS4 as sequencing primers. If sequencing of either the ITS-1 the genus. or ITS-2 region proved difficult, additional sequencing was performed using ITS2 and ITS3 (White et al. 1990) as sequen- Recently, Christensen & Heilmann-Clausen (2013) monograph- cing primers. Sequence contigs were assembled using Se- ed the genus in northern Europe, backed by sequences of the quencher (v. 3.1). Alignment was done with MAFFT (online v. 7) nuclear ribosomal internal transcribed spacer (ITS). The main using the settings G-INS-i (Katoh et al. 2005), with minor manual objective of the present paper is to present the results of the adjustments in Se-Al (Rambaut 1996) for some sequences underlying scientific studies in a coherent form, with the fol- with incomplete ends or internal gaps. Two alignments were lowing specific aims: produced, one containing all sequences in the dataset, and one 1. to evaluate the monophyly of proposed subgenera and sect- based on a reduced dataset containing only one representative